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J. Biol. Chem., Vol. 260, Issue 18, 10153-10160, 08, 1985
ST Olson
The stoichiometry of antithrombin III (AT) inhibition of alpha-thrombin (T)
has been investigated in the presence and absence of heparin as a function
of ionic strength by quantitative titration of enzyme active sites. In
contrast to the ionic strength-independent stoichiometry of 1.0 mol of
AT/mol of T observed in the absence of heparin, the presence of
high-affinity heparin (HAH) resulted in an ionic strength-dependent
increase in the apparent stoichiometry of inhibition from a molar ratio of
1.1 AT/T at an ionic strength of 0.3 to 9.8 mol of AT/T when the ionic
strength was lowered to 0.01. Reduced sodium dodecyl sulfate-
polyacrylamide gel electrophoresis of the reaction products revealed that
the increased AT/T stoichiometry was due to preferential formation of a
specific proteolytically cleaved form of AT that was indistinguishable from
the previously characterized reactive site- cleaved AT (ATM). Using
high-performance liquid chromatography and sodium dodecyl
sulfate-polyacrylamide gel electrophoresis to quantitate ATM, the cleaved
inhibitor was shown to be formed rapidly and concomitant with the stable
thrombin-antithrombin complex (TAT) and quantitatively accounted for the
apparent increase in reaction stoichiometry at low ionic strength in the
presence of HAH. The levels of HAH required to produce maximum ATM were
catalytic at mu greater than or equal to 0.15, but became stoichiometric as
the ionic strength decreased below 0.1. Substantially less ATM was produced
in the presence of low-affinity heparin, while a low molecular weight HAH,
virtually inactive in accelerating T inhibition by AT, was unable to
promote significant ATM formation. These results indicate competition
between substrate and inhibition reactions of AT with T which are affected
by an ionic strength-dependent heparin interaction. A reaction mechanism
accounting for these observations is proposed.
Heparin and ionic strength-dependent conversion of antithrombin III from an inhibitor to a substrate of alpha-thrombin
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